Engineering high-speed allosteric hammerhead ribozymes

Abstract

Full-length hammerhead ribozymes were subjected to in vitro selection to identify variants that are allosterically regulated by theophylline in the presence of a physiologically relevant concentration of Mg(2+). The population of allosteric ribozymes resulting from 15 rounds of in vitro selection yielded variants with observed rate constants (k (obs)) as high as 8 min(-1) in the presence of theophylline and maximal k (obs) increases of up to 285-fold compared to rate constants measured in the absence of effector. The selected ribozymes have kinetic characteristics that are predicted to be sufficient for cellular gene control applications, but do not exhibit any activity in reporter gene assays. The inability of the engineered RNAs to control gene expression suggests that the in vitro and in vivo folding pathways of the RNAs are different. These results provide several key pieces of information that will aid in future efforts to engineer allosteric ribozymes for gene control applications.

Direct selection for ribozyme cleavage activity in cells

“…While the use of allosteric self-cleaving ribozymes to regulate genes in eukaryotic cells has been widely attempted (Link et al 2007;Smolke 2007, 2008) the dynamic range of regulation has been relatively small. For instance, an allosteric self-cleaving ribozyme (Link et al 2007) had a cleavage rate constant in the absence of ligand that was 1/100th that of the wild-type schistosomal HHRz (and similar to that of our selected pools), and thus was likely already high enough to achieve substantial gene knockdown even in the absence of ligand.…”

“…While the use of allosteric self-cleaving ribozymes to regulate genes in eukaryotic cells has been widely attempted (Link et al 2007;Smolke 2007, 2008) the dynamic range of regulation has been relatively small. For instance, an allosteric self-cleaving ribozyme (Link et al 2007) had a cleavage rate constant in the absence of ligand that was 1/100th that of the wild-type schistosomal HHRz (and similar to that of our selected pools), and thus was likely already high enough to achieve substantial gene knockdown even in the absence of ligand. Smolke (2007, 2008) engineered allosteric ribozymes that showed modest (two-to fivefold) activation, and in so doing disrupted loop I/loop II interactions of the TRSV ribozyme, leading to a fivefold reduction in the cleavage (SI Figure 13 of Win and Smolke 2007).…”

Artificial riboswitches for gene expression and replication control of DNA and RNA viruses

“…Clearly, the fact that the constitutively active ribozyme caused potent inhibition of virus replication in all cases indicates that the aptazyme approach should be universally functional with optimized aptazymes. Artificial aptazymes have been mostly developed in vitro and in bacteria, where aptazymes with high induction rates were generated by high-throughput selection (12,13,20,33). However, only a few aptazymes have been reported to be functional in mammalian cells.…”

“…Aptazymes have been extensively developed and studied in vitro, in bacteria, and in yeast (6,12,13,(19)(20)(21)(22). Their application in mammalian systems, however, has been challenging.…”

“…Their application in mammalian systems, however, has been challenging. This is mostly because aptazymes selected in vitro, in bacteria, or in yeast showed loss or strong reduction of regulation when applied in mammalian cells (20,(22)(23)(24). Previous reports on aptazyme applications in mammalian cells were reporter gene studies or studies on regulation of therapeutic gene expression (17,23,25,26).…”